It is well known that both synthetic and natural antioxidants inhibit carcinogenesis and mutagenesis (Shamberger, Volume II, pp. 53-58; Block et al., Am J. Clin. Nutr. 53:270S-282S). Natural antioxidants include ascorbic acid (vitamin C), selenium, oxidized glutathione (GSSG), and reduced glutathione (GSH), which are water-soluble, and the fat-soluble antioxidants .alpha.-carotene, .beta.-carotene (precursor of vitamin A), .alpha.-tocopherol (vitamin E), .gamma.-tocopherol, lycopene and coenzyme Q.sub.10. These antioxidants can be obtained from various food sources, or can be taken as nutritional supplements. Vitamin deficiencies (hypovitaminosis) underlie many human diseases or, conversely, many diseases lead to vitamin deficiencies (Banker et al., Survey of Digestive Diseases 1:203-216, 1983). Hypovitaminosis may result from decreased intake, absorption defects, decreases storage avidity and excessive conversion to metabolites (metabolic destruction).
Randomized clinical trials of vitamin supplementation have shown significant protective effects of .alpha.-tocopherol against prostate cancer, mixtures of retinol/zinc and .beta.-carotene/.alpha.-tocopherol/selenium against stomach cancer; and selenium against total, lung and prostate cancers (Cancer Causes and Control, 8:786-8023 1997).
Antioxidant levels in plasma and tissues have been extensively quantitated. Rose et al. (Biochem. J. 306:101-105, 1995) quantified three water soluble antioxidants, ascorbic acid, reduced glutathione and uric acid, from rat tissue homogenates using high-pressure liquid chromatography. Sinha et al. (Nutrition Res. 17:9, 1409-1415, 1997) examined the stability of ascorbic acid in serum by spectrophotometric determination using 2,4-dinitrophenylhydrazine. Lykkesfeldt et al. (Anal. Biochem. 229:329-335, 1995) determined plasma ascorbic acid levels using HPLC with coulometric detection as a biomarker of oxidative stress. Comstock et al. compared blood antioxidant levels and correlated these levels with the incidence of lung cancer in 258 patients. Among the total group of 258 cases and 515 controls, serum/plasma concentrations were significantly lower among cases than controls for cryptoxanthin, .beta.-carotene, and lutein/zeaxanthin. Modest differences in a protective direction were noted for .alpha.-carotene and ascorbic acid. Stahl et al. (Arch. Biochem. Biophys. 294:173-177, 1992) measured .beta.-carotene and lycopene levels in human serum and tissues. Zhu et al. (Cancer Causes and Control 7:591-595, 1996) analyzed vitamin E levels in breast adipose tissue of breast cancer patients and determined that levels were significantly lower in patients with breast cancer versus benign breast disease. Levine et al. (Proc. Natl. Acad. Sci. U.S.A., 93:3704-3709, 1996) studied steady state plasma and tissue concentrations of vitamin C at seven daily doses from 30 to 2,500 mg. For determination of the Recommended Dietary Allowance (RDA) for Vitamin C, it was concluded that the current RDA of 60 mg daily should be increased to 200-400 mg daily.
The measurement of endogenous antioxidants is of importance because the values obtained may be an indicator of future health. Most methods of determining antioxidant levels focus on serum and adipose tissue. However, because serum levels reflect only those antioxidants which cannot be absorbed, this is not an accurate indication of intracellular levels. Because it is difficult to obtain tissue from an individual for assaying antioxidant levels, this is also not a particularly desirable method. Thus, there is a need for an accurate, convenient method for determining intracellular antioxidant levels to assess the overall oxidative health of an individual and to rule out absorption defects. The present invention provides such a method.